Platelet blockade assay

ABSTRACT

The present invention is an assay for determining glycoprotein IIb/IIIa receptor blockade in whole blood. Agglutinization of small polymeric beads coated with a glycoprotein IIb/IIIa ligand such as fibrinogen results when the beads are contacted with whole blood containing platelets with glycoprotein IIb/IIIa receptors that are not blocked. Failure to agglutinate indicates that blockade of the GPIIb/IIIa receptors has been achieved. In a preferred embodiment, the addition of a thrombin receptor activator results in an assay that is rapid and convenient enough to be performed at the bedside and that results in agglutination of the small polymeric beads within a convenient, known period of time if the glycoprotein IIb/IIIa receptors are not blocked.

CROSS REFERENCES TO RELATED APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 08/315,026,filed Sep. 29, 1994, now U.S. Pat. No. 5,763,199, the disclosure ofwhich is incorporated by reference herein.

BACKGROUND OF THE INVENTION

New agents that block the platelet glycoprotein IIb/IIIa (GPIIb/IIIa)receptor are being developed for use as antithrombotic agents in medicalapplications such as angioplasty and atherectomy. The doses of theseagents required to achieve equivalent antiplatelet effects are likely todiffer as a function of platelet count, glycoprotein IIb/IIIa(GPIIb/IIIa) surface density, and individual variations in drugpharmacokinetics. In fact, in one (Simoons et al. Circulation, 89:596,(1994)) study, a patient with a platelet count greater than 900,000/μl,who received a standard dose of c7E3 Fab had virtually no antithromboticeffect. Since platelet counts within the normal range differ by morethan 2-fold (150,000/μl-350,000/μl), and platelet GPIIb/IIIa density perplatelet also differs by about 2-fold, there may be as much as afour-fold difference in the total number of GPIIb/IIIa receptorsrequiring blockade even within the normal population. Little is knownabout individual differences in the pharmacokinetics of differentGPIIb/IIIa blocking agents, but it is reasonable to expect variationbased on renal function and perhaps other factors that act on lowmolecular weight compounds.

Several assays are currently available for evaluating GPIIb/IIIareceptor blockade, including platelet aggregation, and in the case ofc7E3, either radiolabeled 7E3 binding or flow cytometry. These assaysare time consuming, require special equipment and require extensivestandardization and so are not suitable for bedside monitoring.

It would be desirable, therefore, to have a rapid and simple assay toassess the extent of receptor blockade in an individual. In the settingof angioplasty, it would be desirable to have a GPIIb/IIIa receptorassay conducted at the same time as determination of activated clottingtime (ACT), which is an indicator of extent of heparinization, thatwould indicate when the desired level of blockade had been achieved andthat the patient was ready to undergo the angioplasty. During chronicinfusions, periodic monitoring may also be desirable. Further, incertain circumstances (prior to surgery or an invasive procedure) it maybe desirable to rapidly determine whether the effect of the GPIIb/IIIablocking drug has worn off.

SUMMARY OF THE INVENTION

The present invention relates to a method of determining the level ofresidual unblocked glycoprotein IIb/IIIa receptors in whole blood and,thus, the ability of the individual's platelets to undergo aggregation.Thus, the method refers to a method of determining whether an individualhas reduced ability to form platelet thrombi. The present invention alsorelates to a kit comprising the reagents for carrying out the assay.

The method comprises a first step in which blood is withdrawn from anindividual being assessed for GPIIb/IIIa blockade and is mixed with ananticoagulant. The whole blood/anticoagulant mixture is combined with abuffer which maintains the pH and the salt concentration of the mixtureat levels suitable for platelet aggregation, and with a GPIIb/IIIaligand immobilized on a solid surface. The ligand is immobilized on thesolid surface such that platelet mediated agglutination of the solidsurface in the presence of platelets with unblocked GPIIb/IIIa isdetectable. The resulting mixture is then agitated for a period of timethat is sufficient to allow unblocked platelet GP IIb/IIIa receptors tobind with the GP Ib/IIIa receptor ligand. The absence of agglutinationindicates that the GPIIb/IIIa receptors are blocked. In one aspect thesolid surface is a small polymeric bead on which the ligand isimmobilized at a density to allow the platelets to agglutinate the beadsin the presence of unblocked GPIIb/IIIa receptor. Platelet aggregationpotential is determined by bead agglutination. The absence of beadagglutination indicates that the GPIIb/IIIa receptors are blocked andthe individual has reduced ability to form platelet thrombi. Optionally,a thrombin receptor activating peptide can be added, which causes thebeads to agglutinate more quickly and causes agglutination to occurwithin a defined period of time. The amount of thrombin activatingpeptide can be adjusted so that agglutination occurs within a shorttime, such as two minutes to five minutes if blockade of GPIIb/IIIareceptors is less than 80%. The absence of agglutination indicates thatthe individual will have impaired platelet aggregation which generallycorrelates with prolonged bleeding times and reduced ability to formplatelet thrombi. Alternatively, less thrombin receptor activatingpeptide can be used, such that agglutination will occur within a shortperiod of time, such as two to five minutes, if blockade of GPIIb/IIIareceptors is less than about 50%. This indicates that the individualwill have a normal or near normal bleeding time.

The assay of the present invention has the advantage that it can beconveniently performed at the time of need, such as when a surgicalprocedure is being performed or about to be performed, in a matter ofminutes without the need for expensive equipment or highly trainedmedical personnel. Such an assay has the further advantage that it israpid enough to be used to indicate whether a patient undergoingantiplatelet therapy has achieved a sufficiently high level of GPIIb/IIIa blockade to proceed with a surgical procedure such asangioplasty, in which thrombosis must be avoided. In the event that apatient undergoing antiplatelet therapy requires invasive surgery, theassay is also useful to determine the extent to which the GP IIb/IIIablocking drug is acting and, thus, whether a platelet transfusion isneeded to reverse the effect.

The present invention also relates to a kit that comprises the reagentfor carrying out the assay of the present invention. The presentinvention further relates to a method of diagnosing an individual withGlanzmann thrombasthenia or with the thrombocytopenia using the asset ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

GP IIb/IIIa receptor proteins, which are found on the cell surface ofplatelets, play a central role in thrombosis, i.e., coagulation(clotting of blood. The binding of fibrinogen and perhaps othermacromolecular ligands to GPIIb/IIIa leads to platelet aggregation andis consequently an essential step in the cascade that leads to plateletthrombosis formation and coagulation.

A large enough deficiency in the number of unblocked GPIIb/IIIa receptorproteins in an individual results in a decreased ability of platelets toaggregate, thus resulting in reduced ability to form platelet thrombiand abnormally long bleeding times. For example, blockade of about 80%or more of an individual's GPIIb/IIIa receptors, for example by theadministration of a GPIIb/IIIa receptor blocking agent such asmonoclonal antibody c7E3 Fab, abolishes platelet aggregation andprolongs the bleeding times (period of time in which the individual willbleed from a wound before platelet thrombosis formation stops thebleeding), often to times greater than thirty minutes. Such individualshave an impaired ability to thrombose, i.e., to form blood clots.Certain diseases also result in decreased levels of unblocked GPIIb/IIIareceptors. For example, individuals who are homozygous for Glanzmannthrombasthenia have virtually no GPIIb/IIIa receptor proteins on theirplatelet cell surfaces and also have prolonged bleeding times (Coller etal. Ann. N.Y. Acad. Sci 614:193 (1993). In addition, individuals who areseverely thrombocytopenic, i.e., have platelet counts below 100,000platelets/μl, have similar prolonged bleeding times. However, plateletaggregation is unaffected or minimally affected in individuals untilabout 40-50% of GPIIb/IIIa receptors are blocked and bleeding times arenormal until as many as about 60% of GPIIb/IIIa receptors are blocked.Levels of GPIIb/IIIa blockade up to about 80% result in mild to moderateabnormalities in platelet aggregation and bleeding times (Coller et al.Ann. N.Y. Acad. Sci 614:193 (1993)). Consistent with these figures isthe observation that individuals who are heterozygous for Glanzmannthrombasthenia and who have only 50-60% of the normal numbers ofGPIIb/IIIa receptors show normal bleeding times. Consequently, theability of the blood of an individual to undergo platelet aggregationand coagulate is dependent on the number of unblocked GPIIb/IIIareceptors.

One embodiment of the present invention is a method of assessing whetheran individual will have severely impaired ability to form plateletthrombi or impaired platelet aggregation due to blockade of about 80% ormore of the GPIIb/IIIa receptors or to a similar deficiency in thenumber of GPIIb/IIIa receptors. An individual with a blockade of 80% ormore of the GPIIb/IIIa receptors will have prolonged bleeding times,i.e. bleeding times of greater than about thirty minutes, an impairedability to form platelet thrombi and impaired platelet aggregation. Themethod comprises determining whether greater than 80% of the GP IIb/IIIareceptors are blocked in whole blood. Blockade of 80% or more of GPIIb/IIIa receptors in whole blood is determined by withdrawing bloodfrom an individual being assessed for GPIIb/IIIa blockade and mixing theblood with an anticoagulant. The whole blood/anticoagulant mixture isthen combined with 1) a buffer that is capable of maintaining the pH andsalt concentrations of the blood at levels suitable for plateletaggregation and 2) a GP IIb/IIIa ligand immobilized on a solid surfacesuch that platelet mediated agglutination of the solid surface isdetectable when the solid surface is combined with platelets havingunblocked GPIIb/IIIa receptors. The resulting mixture is then agitatedfor a period of time that is sufficient to allow unblocked platelet GPIIb/IIIa receptors to bind with the GP IIb/IIIa receptor ligand. Theabsence of platelet aggregation indicates that more than about 80% ofthe GPIIb/IIIa receptors are blocked. The absence of platelet mediatedagglutination of the solid surface also indicates that the individualwill have reduced platelet aggregation, a prolonged bleeding time andreduced ability to form thrombi. Platelet mediated agglutination of thesolid surface indicates that less than about 80% of the GPIIb/IIIareceptors are blocked. Preferably, the solid surface is a smallpolymeric bead. The GPIIb/IIIa ligand must be immobilized at asufficient density on the surface of the beads so that the beads canagglutinate in the presence of platelets with GPIIb/IIIa receptors thatare not blocked. The absence of agglutination indicates blockade orabsence of GPIIb/IIIa receptors.

As used herein, GPIIb/IIIa receptors are blocked when a drug, peptide,monoclonal antibody or small organic molecule binds to, complexes withor interacts with the GPIIb/IIIa receptor such that a natural ligand ofthe GPIIb/IIIa receptor, for example fibrinogen, cannot bind to theGPIIb/IIIa receptor.

Blood can be drawn from the individual being assessed for GPIIb/IIIareceptor blockade by any number of known techniques. Preferably, theblood is drawn into a Vacutainer or other closed tube to protect theindividual who is drawing the blood from blood-borne infectious agentssuch as the hepatitis B virus or the HIV virus. The whole blood is thenmixed with an anticoagulant. This can be carried out, for example, bydrawing the blood into a tube containing anticoagulant or by addinganticoagulant to drawn blood. A sufficient amount of anticoagulant isused to prevent coagulation of the whole blood. Suitable anticoagulantsinclude citrate, oxalate, ACD-A, heparin, hirudin or other antithrombinagent. A preferred anticoagulant is citrate. The final citrateconcentration in the whole blood ranges from 0.3% to about 0.5% of theblood, preferably about 0.4%. Whole blood to which a sufficient amountof anticoagulant has been added to prevent coagulation is referred to as"anticoagulated blood".

The anticoagulated blood is mixed in an assay tube with a buffer and asuitable amount of small polymeric beads on whose surface a ligand thatbinds to the platelet GPIIb/IIIa receptor has been immobilized toproduce an anticoagulated blood-bead mixture. The ligand is immobilizedat a density sufficient to allow the beads to agglutinate in thepresence of platelets with GPIIb/IIIa receptors that are not blocked.

A suitable buffer is capable of maintaining the pH and saltconcentration of the blood within a range suitable for plateletaggregation. Suitable pH levels are about 6.6-8.2 and is preferablyabout 7.4. The buffer may be added to the blood sample either as asolution or solely as the buffering composition and salts. A suitablebuffer comprises a concentration of a buffering composition such asHEPES, Tris or others that maintain the pH of the blood within the rangesuitable for platelet aggregation, as defined above. HEPES is apreferred buffer and is present generally at a concentration of 0.01M. Asuitable buffer also maintains the salt concentration of the bloodwithin a range suitable for platelet aggregation. Consequently, thebuffer contains a concentration of one or more salts, such as NaCl, thatmaintain the electrolytic balance of the blood within a range suitablefor platelet aggregation. Suitable concentrations of NaCl in the bufferare between 0.10M and 0.20M, but typically 0.15M. In addition, saltssuch as CaCl₂ are required for binding between the GPIIb/IIIa receptorand the GPIIb/IIIa receptor ligand such as fibrinogen. Suitable bufferconcentrations of CaCl₂ range between about 0.1 mM and about 1.0 mM, andare preferably about 0.5 mM.

A GPIIb/IIIa ligand is a small organic molecule, polypeptide, protein,monoclonal antibody or nucleic acid that binds, complexes or interactswith GPIIb/IIIa receptors on the platelet surface. The platelet mediatedagglutination of the solid surface results when the GPIIb/IIIa receptorson the surface of platelets bind, complex or interact with GPIIb/IIIaligands bound to the solid surfaces. Suitable GPIIb/IIIa ligands includefibrinogen, monoclonal antibody lOES (Coller et al. J. Clin. Invest.72:325 (1983), deposited with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md., Accession No. 1006088,monoclonal antibody c7E3 (The EPIC Investigators, N.E. Journal of Med.,330:956 (1994), other monoclonal antibodies, von Willebrand factor,fibronectin, vitronectin and ligands that comprise anarginine-glycine-aspartic (RGD) acid sequence or other peptides orpeptidomimetics that mimic this sequence (Cook et al. Drugs of theFuture 19:135 (1994)). The RGD functional equivalent ligands includegamma chain peptides, peptidomimetic and cyclic peptides with anactivity that is about the same as an RGD ligand.

A solid surface is any surface onto which a GPIIb/IIIa ligand can beimmobilized such that platelet mediated agglutination of the solidsurface is detectable when the solid surface is combined with plateletshaving unblocked GPIIb/IIIa receptors. Typically, the ligand isimmobilized on the surface at a density such that platelet mediatedagglutination of the solid surface can occur by binding of the plateletGPIIb/IIIa receptor with the immobilized ligand. The ligand isimmobilized on the solid surface by covalent binding, complexing oradsorption, such that the ligand is capable of binding to GPIIb/IIIareceptors. Suitable solid surfaces include glass (for example, glassparticles or test tubes), plastic, or small polymeric beads. Smallpolymeric beads are preferred. The detection of platelet mediatedagglutination of the solid surface can be accomplished visually or bymeans of a magnification aid. Preferably, platelet binding to the solidsurface is detected by observing some change in the solid surface whichoccurs as a consequence of platelet binding, for example, agglutinationof small polymeric beads or plastic or glass microparticles.

A small polymeric bead, as used herein is any polymeric microparticle aGPIIb/IIIa receptor ligand can be covalently bound to or adsorbed on.The polymeric microparticles can be virtually any shape, but aregenerally spherical with uniform diameters ranging from about 0.1 μm toabout 10 μm in diameter. Preferred diameters are from about 1.0 μm toabout 6.0 μm in diameter. For example, the polymeric microparticle maybe polymerized acrylonitrile 1-3 μm beads with N-hydroxysuccinimideester groups on their surface (e.g., Matrex 102 beads from AmiconCorporation) (Coller, Blood, 55:169 (1980)). The N-hydroxysuccinimideester groups allow coupling of the N-terminus of the peptide, protein ormonoclonal antibody to the surface of the bead (see example 2).Alternatively, the microparticle can be carboxylated polystyrene 3-6 μmbeads (Polysciences Inc.). The surface carboxyl groups of this bead canbe coupled to the N-terminus of the protein, peptide or monoclonalantibody by means of a carbodiimide coupling (see Example 1). The beadsmay be colored to render the results of the agglutination reactioneasier to interpret.

In carrying out the method of the present invention, 10 μl-200 μl ofanticoagulated blood, 10 μl-200 μl of buffer solution and 1 μl-50 μl ofsmall beads are mixed in the second step of the present invention. Inone embodiment, about 50 μl of anticoagulated blood, about 50 μl ofbuffer solution, and about 5 μl of small beads are used. Alternatively,the mixture can be scaled up or down, as long as the ratio of thecomponents used remain within the limits described above.

As discussed previously, the GPIIb/IIIa receptor ligand can also be aligand comprising an arginineglycine-aspartic acid sequence, or mimicthereof, which is covalently bound to the solid surface through asuitable spacer. Examples of suitable ligands are disclosed in Beer etal., Blood 79:117 (1992), the contents of which are incorporated intheir entirety herein by reference. Suitable GPIIb/IIIa receptor ligandsinclude the peptide (glycine)_(n) -arginine-glycine-aspartic wherein nis an integer from 2-20 (SEQ ID NO:9). The polyglycine portion of theligand is the spacer and is covalently bound to the surface of thepolymeric bead at the N-terminal amino group. While the RGD sequence mayparticipate in the binding to platelets, a gamma chain sequence forminga molecular mimic of the RGD sequence may be more important in thebinding of fibrinogen to platelets (Coller, Platelet Morphology,Biochemistry, and Function, 1175). Divalent cations such as calcium,magnesium, and manganese are required for the binding of all GPIIb/IIIaligands, and each ligand differs subtly with respect to the preferredcation. The competence of activating agents also varies among ligands(Id. at 1175, 76). Optionally, an additional amino acid or oligopeptidewhich does not significantly interfere with the binding ofarginine-glycine-aspartic acid to the GPIIb/IIIa receptor may be boundto the C-terminus of aspartic acid by means of a peptide bond. In oneembodiment, the GPIIb/IIIa receptor ligand comprises (Glycine)₉₋₁₁-arginine-glycine-aspartic acid-phenylalanine (SEQ ID NO:10).Alternatively, the spacer portion of the ligand can comprise any moietywhich causes the arginine-glycine-aspartic acid sequence to extend outfrom the surface of the microparticle sufficiently to allow bindingbetween the ligand and GPIIb/IIIa receptors on the surface of plateletsand does not significantly interfere with the ability ofarginine-glycine-aspartic acid to bind with GPIIb/IIIa. Examples ofsuitable moieties include alkyl groups and polyglycol groups.

The mixture produced is agitated for a period of time sufficient toallow the platelet GPIIb/IIIa receptors to bind with the GPIIb/IIIareceptor ligand on the small polymeric beads and agglutination of thebeads to occur. Agitation is preferably supplied mechanically, forexample by placing the assay tube on a rocker. The assay tube can berocked for up to twenty minutes at 5-40 cpm, but preferably about twominutes at about 20 cpm. Alternatively, the mixture produced can beagitated manually, for example by placing the mixture on a glass plateand rotating or by mixing with stick. Manual agitation can be continuedfor as long as twenty minutes, but preferably for about 2-3 minutes.

Subsequently, the mixture is assessed to determine whether agglutinationof the beads has occurred and, optionally, the extent of theagglutination. Lack of agglutination of the beads indicates that theGPIIb/IIIa receptors on the platelets in the sample are blocked, i.e.,greater than about 80% of the GPIIb/IIIa receptors are blocked. Lack ofagglutination also indicates that the individual being assessed willhave prolonged bleeding times due to about 80% or greater GPIIb/IIIablockade.

Agglutination of the beads indicates that less than about 80% ofGPIIb/IIIa receptors on the platelets in the sample are blocked. Theextent of agglutination is indicative of the level of blocked GPIIb/IIIareceptors. A normal level of agglutination indicates that less thanabout 40-50% of the individual's GPIIb/IIIa receptors are blocked andthat the individual will have a normal or near-normal bleeding time. Alevel of agglutination that is less than normal indicates that betweenabout 50% and about 80% of the individual's GPIIb/IIIa receptors areblocked, and that the individual will have a mild to moderatelyprolonged bleeding time, i.e., a bleeding time that is longer thannormal but less than about thirty minutes. Whether agglutination is lessthan normal is determined by comparing the amount of agglutination fromthe blood of an individual being assessed with the amount ofagglutination from a standard or control. The standard or control is theamount of agglutination that results from the blood of an individualwith a normal platelet count (150,000-350,000 platelets/μl) whoseGPIIb/IIIa receptors are more than about 60% unblocked. The standard orcontrol can be predetermined or run simultaneously.

Agglutination is determined visually by whether the beads clump, whichis indicative of agglutination, or whether they remain suspended in thesolution, which is indicative of the absence of agglutination.Optionally, the beads can be colored to aid in visualizingagglutination, or clumping, of the beads. The level of agglutination isalso determined visually by comparing whether there is less clumping inthe solution compared with the control or standard.

In the method of the present invention, a thrombin receptor activatingpeptide is optionally combined with the mixture of anticoagulated blood,buffer and small polymeric beads to which a GPIIb/IIIa receptor ligandis bound or adsorbed. The thrombin receptor is a transmembrane proteinthat is present in platelets (Vu et al. Cell 64:1057 (1992)). A thrombinreceptor activator, as defined herein, is a peptide, protein, antibodyor small organic molecule that induces platelet activation via thethrombin receptor, i.e., which increases the rate of agglutination whenplatelets whose GPIIb/IIIa receptors are not blocked when the plateletsare combined with a GPIIb/IIIa receptor ligand bound to solid surfaces.A suitable peptide is any peptide of appropriate sequence and size toactivate platelets, as described above. The peptide can comprisethrombin, or a portion thereof, such that the amino acid sequence of thepeptide or peptide mimic result in activation of the platelets. Vu etal. identified the amino acid sequence of the thrombin receptor andproposed a mechanism of thrombin receptor activation. Thrombin cleavesthe thrombin receptor protein, releasing a short receptor fragment andleaving a new amino terminal peptide on the platelet surface. The newamino terminal peptide activates the receptor by functioning as atethered ligand that interacts with another region of the receptor toinduce activation signals. A fourteen amino acid peptide (T-14) (SEQ IDNO: 1) corresponding to the new N-terminus of the cleaved receptorprotein is capable of aggregating platelets directly without priorthrombin cleavage (Vu et al.). However, the entire peptide is notrequired for activity because an eleven amino acid peptide (T-11) (SEQI.D. NO: 2) lacking the three C-terminal amino acids of T-14 is twice aspotent as T-14 (see Coller et al. Biochemistry 31:11713 (1992), thecontents of which are hereby incorporated by reference into thisapplication in their entirety). A peptide comprising the first five orsix amino acids has also been shown to be active. (Vassallo et al., J.Biol. Chem. 267: 6081 (1992), Hui et al., Biochem. Biophys. Res. Commun.184:790 (1992), Sabo et al., Biochem. Biophys.. Res. Commun. 188:604(1992) and Scarborough et al., J. Biol. Chem. 267:13146 (1992)). Infact, T-6 (SEQ ID NO: 4) has been shown to be more active than thelonger T-11 amino acid of SEQ ID NO:2, with the latter having ˜20% ofthe platelet activation activity of T-6. (U.S. Pat. No. 5,455,228(Coller)).

An important application of the assay of the present invention is forrapid monitoring of an individual during a medical procedure or todetermine whether an individual is ready to undergo a medical procedure.This type of monitoring is referred to as "bedside monitoring." Forexample, monoclonal antibody c7E3 is given prior to angioplasty oratherectomy in high-risk clinical situations to reduce the risk ofischemic complications (The EPIC Investigators, N.E. J. of Medicine 330:956 (1994)). Monoclonal antibody c7E3 acts by blockading the GPIIb/IIIareceptor. Because GPIIb/IIIa blockade inhibits platelet thrombusformation, it is important that blockade of this receptor be reversedprior to invasive surgery in the event that invasive surgery is neededfollowing c7E3 Fab injection. It is advantageous to have an assay thataccurately and rapidly determines the extent to which GPIIb/IIIaplatelet receptors in the patient's blood are blocked and, thus, thelikelihood excessive bleeding will occur. When GPIIb/IIIa blockade isachieved, the patient is ready to undergo the procedure. The addition ofa thrombin-activating peptide or other thrombin-activating agent has theadvantage of inducing platelet activation, thereby increasing the rateat which the beads of the assay of the present invention agglutinateand, thus, decreasing the time required for the assay to be carried out.

The N-terminal serine group of the thrombin receptor activatingpeptides, e.g., T-11, T-6, and T-14 (SEQ ID NO:7), is essential to thesepeptides' ability to induce platelet aggregation. This is based on theobservation that acetylation of the N-terminal serine of T-11 results inloss of aggregating ability. In addition, T-11 and T14 lose theirability to induce aggregation when incubated in plasma, becauseaminopeptidase M, present in plasma, cleaves the N-terminal. Significantlosses of platelet aggregating ability can occur in as little as tenminutes (Coller et al., Biochemistry 31:11713 (1992)). The plateletaggregating activity of T-6 (SEQ ID NO:4) was shown to decrease within15 minutes of incubation in plasma and was nearly lost after 60 minutes(U.S. Pat. No. 5,455,228 (Coller)). The presence of aminopeptidase M inwhole blood can result in variability in the amount of time required foragglutination of the beads in the assay of the present invention.

The variability in the time required for agglutination can be avoided bycarrying out the present method under conditions wherein the cleavage ofthe N-terminal serine of the thrombin receptor activating peptide issuppressed. This can be accomplished by employing a thrombin receptoractivating peptide that is resistant to degradation by aminopeptidase M.A resistant thrombin receptor activating peptide should have theadditional property of retaining sufficient activity such that when theresistant thrombin receptor activating peptide is added to the assay ofthe claimed invention, as described above, agglutination is rapidenough, when the platelet GPIIb/IIIa receptors are not blocked, that theassay can be used for bedside monitoring. An assay suitable for bedsidemonitoring results in agglutination in less than ten minutes, butpreferably in less than five minutes. A preferred thrombin receptoractivating peptide that is resistant to degradation by aminopeptidase Mis racemic isoSer-Phe-Leu-Leu-Arg-Asn (SEQ ID NO: 3). This peptide ishereinafter referred to as T-6'. Isoserine is an isomer of serine havingthe formula NH₂ CH₂ CHOHCOOH. The C-2 position of isoserine is chiral,and racemic isoserine is a mixture of D and L isomers. One or both ofthe isomers is active. T-6' retains 15-20% of the activity of the sixamino acid thrombin receptor activating peptide Ser-Phe-Leu-Leu-Arg-Asn(SEQ ID NO. 4) while showing no significant loss of activity after twohours of incubation with platelet poor plasma (see Coller and Prestwich,U.S. Pat. No. 5,455,228, and Coller et al., J. Biol. Chem. 268:20741(1993)), the contents of which are hereby expressly incorporated intheir entirety into this application by reference). Other suitablethrombin receptor activating peptides that are resistant toaminopeptidase M include peptides comprising an N-terminus having theamino acid sequence of T-6', such that the peptide is resistant toaminopeptidase M degradation and retains sufficient platelet activatingactivity, as described above. Examples include analogues of T-11 (SEQ IDNO: 5) and T-14 (SEQ ID NO: 6), wherein the N-terminal serine of T-11 orT-14 is substituted with iso-serine. Although Applicant does not wish tobe bound by any particular mechanism, it is believed that these peptidesare resistant to inactivation by aminopeptidase M because isoserine isnot cleaved from the N-terminus. Specific requirements for each group ofthe aminopeptidase M resistant peptides have been determined. R₁ may beisoserine, 2,3-diaminopropanoate or 2,3-dihydroxypropanoate. R₂ may beany hydrophobic amino acid residue having a cyclic or aromatic group.Preferred amino acids for R₂ are phenylalanine, p-benzoylphenylalanine,p-fluorophenylalanine, and p-methylphenylalanine. R₃ may be anyhydrophobic amino acid such as alanine, phenylalanine, glycine,isoleucine, leucine, methionine, proline, serine, threonine, tryptophanand valine. Preferred are leucine, phenylalanine, and alanine, and mostpreferred is leucine. R₄ may be any hydrophobic amino acid such as thoselisted for R₃. Preferred are leucine, and alanine, and most preferred isleucine. Each of R₅ through R₁₄ may be absent or, if present, is anL-amino acid with the terminal amino acid being a carboxy orcarboxamide, or a moiety that enhances cell penetrating ability of thepolypeptide. Such moieties are known in the art and include lipids,steroids, long chain amine's such as decylamine, and sugars such asconjugates of glucosamine, glucuronic acid, etc. Preferred R₅ aminoacids are arginine and alanine, and most preferred is arginine, andpreferred R₆ is asparagine. The present invention also encompasses otherthrombin. receptor activating peptides such as analogues of T-14, T-11,the 6-mer of T-14 and the 5-mer of T-14 (SEQ ID NO:8) in which theN-terminal serine is substituted with a moiety, preferably an aminoacid, that results in resistance of the peptide to cleavage byaminopeptidase M. Also encompassed are peptide stereochemical varietieswith resistance to aminopeptidase M. While T-11 with an N-terminalserine group in the D-configuration has only minimal aggregatingactivity, it has been shown to retain more than half its aggregatingactivity when incubated in plasma for 1 hour (U.S. Pat. 5,455,228(Coller)). Suitable thrombin receptor activating peptides can beprepared by peptide synthesis, according to methods known to thoseskilled in the art (See Example 3).

Alternatively, the variability can be avoided by including an inhibitorof aminopeptidase M in the assay. A suitable inhibitor of aminopeptidaseM includes amastatin, which has been shown to enhance plateletaggregation in the presence of aminopeptidase M (Coller et al.,Biochemistry 31:11713 (1992)). Acetylation of the N-terminus of thethrombin receptor peptide ligand is the traditional method for producinga peptide that resists cleavage by aminopetidase M, but acetylation ofthis ligand eliminates receptor activator activity. Otherplatelet-activators can be used in place of the thrombin receptoractivating peptides described above. For example, adenosine diphosphate,collagen, ristocetin, botrocetin, epinephrine, arachidonic acid and itsmetabolites including thromboxane A₂, platelet activating factor,plasmin, serotonin, vasopressin, tissue plasminogen activator,streptokinase and immune complexes can be added, alone or in combinationwith other platelet activators, to increase the rate of agglutination ofthe beads in the assay of the present invention. Additionally,application of high levels of shear stress, and artificial surfaces suchas those used clinically for prosthetic materials can also activateplatelets (Coller, Platelet Morphology, Biochemistry, and Function,1185).

A preferred embodiment of the present invention is based on thediscovery that as a consequence of platelet activation by a thrombinreceptor activating peptide, such as T-6', the time period required foragglutination of the small beads is rapid enough for bedside monitoringand is reproducible when the amounts of reagents and the percentage ofblocked GPIIb/IIIa receptors in the blood being tested remain constant.For example, 70 μl of small beads bound with fibrinogen in the presenceof 100 μl buffer (0.01M HEPES, 0.15M, NaCl, 0.5 mM CaCl2), 70 μlanticoagulated whole blood and 5-10 μl T-6' (see Example 5) willagglutinate in two minutes if high grade GPIIb/IIIa blockade has notbeen achieved, i.e. when less than 80% of the GPIIb/IIIa receptors areblocked. The concentration of T-6' in this assay can generally rangefrom between about 2-5 μM, which allows variations in the amounts of theother reagents. The skilled artisan can readily determine suchvariations. This provides a quick and simple-assay that can be performedat the bedside to determine when a patient undergoing antiplatelettherapy has achieved GPIIb/IIIa blockade.

A bedside assay for GPIIb/IIIa can be used in conjunction with a varietyof other medical procedures, for which levels of GPIIb/IIIa blockade maydiffer. For example, platelet transfusions may be needed prior toinvasive surgery in order to prevent excessive bleeding if GPIIb/IIIablockade is greater than 50% (Coller et al. N.Y. Acad. Sci., 614:193(1993)). Determining whether GPIIb/IIIa receptor blockade has returnedto normal levels is particularly important if invasive surgery is neededimmediately after a procedure such as angioplasty or atherectomy, inwhich the GPIIb/IIIa blockade has been elevated by antiplatelet therapy.Another embodiment refers to an assay to determine when 50% or less ofthe GPII/IIIa receptors of an individual are blocked. The samequantities of reagents are used as with the assay for determining 80%blockade, as discussed above, except that the concentration ofisoSer-Phe-Leu-Leu-Arg-Asn is reduced to about 0.2-1 μM. Agglutinationwithin two minutes indicates that the individual will have a normal ornear normal bleeding time. Less than about 50% of the GPIIb/IIIareceptors are blocked in this individual.

Assays that are indicative of different levels of GPIIb/IIIa blockadecan be readily determined by one of ordinary skill in the art. Wholeblood is drawn from an individual with a normal platelet count,(150,000-300,000 platelets/μl) in whom a known percentage of GPIIb/IIIareceptors are blocked. The amounts of reagents used in the assay, i.e.anticoagulated blood with the known percentage of GPIIb/IIIa blockade,buffer, small beads and T-6' or other platelet activator can be varieduntil a ratio of reagents is found that results in agglutination withina convenient period of time. Generally, it is most convenient to varythe amount of thrombin receptor activator in order to obtain an assay inwhich agglutinization occurs within a desired period of time for acertain level of GPIIb/IIIa blockade. The percentage of GPIIb/IIIareceptors in the blood that are blocked can be determined by methodsknown to those skilled in the art, e.g. radiolabeled 7E3 binding (Colleret al., N. Y. Acad. Sci., 614:193 (1993)) or flow cytometry (Coller etal. Blood 74:182, (1989)). This ratio of reagents can be used in theassay of the present invention to test the extent of GPIIb/IIIa blockadein a patient in whom the extent of GPIIb/IIIa blockade is unknown.Agglutination of the beads within the desired period of time indicatesthat extent of GPIIb/IIIa blockade is less than or equal to theindicated percentage. Failure of the beads to agglutinate indicates thatGPIIb/IIIa blockade equals or exceeds the indicated percentage. If theplatelet count is abnormally low, there may be no agglutination, evenwith there being relatively little receptor blockade. It is necessary toperform a platelet count to know that it is normal (150,000-350,000/μl).

A kit is provided herein, in which the reagents necessary for carryingout the assay of the present invention are provided. The kit comprises ablood vial, a buffer that maintains the pH and salt concentration of theblood sample assessed within ranges suitable for platelet mediatedagglutination of the solid surface and small polymeric beads coated withplatelet GP IIb/IIIa receptor ligand. The buffer can be in solution, orcan consist solely of the buffering composition and salts. The bufferingcomposition and salts may be added directly to the blood sample to avoiddilution of the blood, or added as a solution after being combined witha known amount of water to give the desired buffer solution. optionally,the kit can also comprise an anticoagulant. In one embodiment, thepreferred buffer is HEPES; the anticoagulant is citrate; a GPIIb/IIIareceptor ligand is fibrinogen; small polymeric beads arepolyacrylonitrile or carboxylated polystyrene in which a peptideGPIIb/IIIa receptor ligand, such as fibrinogen, is covalently bonded tothe bead surface by means of a covalent bond between the N-terminus ofthe peptide and an N-hydroxysuccinimide or carboxylate group on the beadsurface.

In a further embodiment, the kit additionally comprises a plateletactivator, such as a thrombin receptor activating peptide. For example,the thrombin receptor activating peptide is isoSer-Phe-Leu-Leu-Arg-Asp.If the thrombin receptor activating peptide is not resistant todegradation by aminopeptidase M, the kit can additionally comprise aninhibitor of aminopeptidase M, such as amastatin.

As discussed above, it is advantageous for certain applications of thepresent invention that the assay of the present invention be used forbedside monitoring and that the assay indicate whether a certain levelof GPIIb/IIIa receptor blockade has been achieved. As discussed earlier,the relative amounts of reagents can be adjusted so that agglutinationof the beads results within a desired period of time if the desiredlevel of GPIIb/IIIa receptor blockade is not achieved. A kit to be usedin this context generally contains the quantity of buffer, thrombinreceptor activating peptide, such as isoSer-Phe-Leu-Leu-Arg-Asp, andGPIIb/IIIa ligand-coated polymeric beads such that agglutination of thebeads will result within a known period of time suitable for bedsidemonitoring when mixed with a pre-determined amount of anticoagulatedwhole blood in which the desired level of GPIIb/IIIa receptor blockadehas not been achieved. For example, if the method is to be carried outin approximately two minutes and it is to be determined whether thelevel of GPIIb/IIIa blockade is 80% or less, the kit comprises 100 partsby volume buffer to 10 parts by volume beads to 5-10 parts by volumeisoSer-Phe-LeuLeu-Arg-Asp. In another aspect, the desired period of timeis two minutes and the level of GPIIb/IIIa- blockade is 50% or less.

Another embodiment of the present invention refers to a method ofdiagnosing individuals with bleeding abnormalities due to reduced levelsof unblocked GPIIb/IIIa receptors. For example, individuals who arehomozygous for Glanzmann thrombasthenia have virtually no functionalGPIIb/IIIa receptors and consequently have prolonged bleeding times.Individuals who are heterozygous for Glanzmann thrombasthenia haves50-60% of the normal number of GPIIb/IIIa receptors, but show normalbleeding times (Coller et al. Ann. N.Y. Acad. Sci. 614:193 (1993)).Individuals with platelet levels below about 100,000 platelets/μl areseverely thrombocytopenic and also have prolonged bleeding times.Consequently, when the blood of an individual who has not beenadministered an agent that blocks GPIIb/IIIa receptor and who ishomozygous for Glanzmann thrombasthenia or is severely thrombocytopenicor has other platelet function defects is tested for "blockade" ofGPIIb/IIIa receptors by the assay of the present invention, noagglutination of the beads will be observed. The method of detecting anindividual who is homozygous for Glanzmann thrombasthenia, or has otherplatelet function defects, or is thrombocytopenic comprises testing theblood of the individual for GPIIb/IIIa blockade by the assay describedherein. The blood of the individual should be tested when the individualhas not been administered an agent, e.g., an agent used for antiplatelettherapy, that blocks GPIIb/IIIa receptors. The absence of agglutinationof the beads indicates that the individual should be tested further forGlanzmann thrombasthenia, other platelet function defects orthrombocytopenia.

The invention is further illustrated by the following examples, whichare not intended to be limiting in any way.

EXEMPLIFICATION EXAMPLE 1 Preparation of Fibrinogen Coated CarboxylatedPolystyrene Beads

0.5 ml of 2.5% carboxylated microparticles obtained from PolysciencesIncorporated were placed into an Eppendorf centrifuge tube having acapacity of 1.5-1.9 ml. A sufficient amount of 0.1M carbonate buffer, pH9.6, was added to fill the tubes, which was then centrifuged for 56minutes in a micro-centrifuge. The supernatant was carefully removedusing a pasteur pipette and discarded. The pellet was then resuspendedin carbonate buffer. To resuspend the pellet, the tube was filledhalfway, capped, vortexed and then filled to capacity. The tube was thenrecentrifuged for 5-6 minutes and the supernatant removed and discarded.The pellet was then resuspended in 0.02M sodium phosphate buffer, pH4.5, and centrifuged for 5-6 minutes. The supernatant was then removedand discarded. The resuspension in phosphate buffer and centrifugationwas repeated two more times, after which the supernatant was removed anddiscarded.

A 2% solution of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimidehydrochloride dissolved in 0.02M sodium phosphate (2% carbodiimide)buffer was freshly prepared within 15 minutes of using. 0.625 ml of this2% carbodiimide solution was added dropwise to the pellet. The resultingsuspension was mixed for 3-4 hours at room temperature using anend-to-end mixture. The suspension was then centrifuged for 5-6 minutesand the supernatant was removed and discarded. The resulting pellet wasresuspended in 0.02M phosphate buffer and centrifuged for 5-6 minutes.The supernatant was then removed and discarded. This process ofresuspending in phosphate buffer and centrifuging was repeated two moretimes.

The resulting pellet was suspended in 0.2M borate buffer, pH 8.5. 300 μgof fibrinogen was added to the suspension, which was then gently mixedovernight at room temperature on an end-to-end mixer. 50 μl of 0.5Methanolamine was then added and the suspension mixed for 30 minutes.

The suspension was then centrifuged for 10 minutes, and the supernatantwas removed and discarded. The pellet was then resuspended in 1 ml of 10mg per milliliter BSA solution in borate buffer. The mixture was capped,vortexed and then gently mixed for 30 minutes at room temperature. Aftercentrifuging for 5-6 minutes the supernatant is removed and discarded.This process of resuspending the pellet in BSA solution and centrifugingwas repeated one time. The supernatant was then removed and discarded,and the pellet resuspended in 0.5 ml of PBS, pH 7.4, containing 10 mgper milliliter BSA, 5% glycerol, and 0.1% NaN₃, and stored at 4° C.

The extent of coupling was monitored by immunologic fibrinogendetermination, optical density at 280 nm and or protein assays (Lowry orBio-Rad Protein Assay).

EXAMPLE 2 Covalent Coupling of Fibrinogen to Polymerized Acrylonitrile

Solid beads of polymerized acrylonitrile, primarily 1-3 μm in diameter,with free carboxyl groups and N-hydroxysuccinimide groups on theirsurface (Matrex 102, Amicon Corporation) come packed in dioxane toprevent hydrolysis of the esters. One milliliter of a well mixedsuspension (containing 67 mg of beads) was centrifuged at 10,000 g for 1minute, the supernatant dioxane removed, and the beads rapidly washedtwice with 1.2 ml of 0.01M Na acetate, pH 6. The dried, washed beadswere then resuspended in 1.5 ml of either fibrinogen or albumin (2mg/ml) solution in 0.03M sodium citrate, pH 7.5, and allowed to rockovernight at 4° C. In some experiments, a smaller volume of a moredilute protein solution (0.7 mg/ml) was employed, but the same ratio oftotal protein to beads (0.045) was always maintained. The extent ofcoupling was monitored by immunologic fibrinogen determination, opticaldensity at 280 nm and/or protein assays (Lowry or Bio-Rad Protein Assay)on the postcoupling supernatant after dialysis (to remove theinterfering chromophore released by the reaction). In 8 experiments, thefibrinogen coupled with 88%-100% efficiency, whereas in 4 experiments,the albumin coupled at 17%-27% efficiency. Since complete coupling offibrinogen required only ˜0.1% of the total reactive sites, the reactionproceeded very rapidly and was complete in less than 7 minutes.Calculations based on the manufacturer's estimate of the surface area ofthe beads (6 sq. m/g) indicate that there is approximately 1 molecule offibrinogen every 8000 square Å of bead surface, which translates intoapproximately 1 molecule of fibrinogen every 90 Å. On average, therewere ˜500, 000 fibrinogen molecules coupled to each bead. When adjustedfor the difference in molecular weight, virtually the same number ofalbumin molecules were bound to the beads as were fibrinogen molecules.After the coupling procedure was completed, the beads were washedextensively with 0.15M NaCl, 0.01M Tris-HCL, 0.05% azide, pH 7.4 untilthe supernatant buffer's absorbance at 280 nm reached baseline so as toremove the products of the reaction and any nonspecifically absorbedfibrinogen. Both the fibrinogen and albumin beads were then resuspendedin equal volumes of buffer (2-4 ml), giving a particle count of˜250,000/μl. To assess the amount of fibrinogen that adsorbed to thebeads instead of being covalently coupled, beads were suspended in asolution of 3% SDS 8M urea, 0.02M NaPO for 30 minutes at 37° C., and theprotein content in the solution determined. Less than 2' of the boundprotein could be eluted in this way.

EXAMPLE 3 Synthesis of Thrombin Receptor Activating Peptides

This example describes synthesis of thrombin receptor activatingpeptides, T-11, T-14 and T-6'. The methods described can be used toproduce other thrombin receptor activating peptides. T-14 and T-11 wereprepared on an automated peptide synthesizer (Applied Biosystems 430A;Foster° City, Calif.) using t-Boc chemistry, 4-methylbenzhydrylamineresins and N-methylpyrrolidone as the coupling solvent as previouslydescribed (Beer et al., Blood, 79: 117 (1992)). The protecting groupswere β-benzyl ester for aspartic acid, benzyl for serine,2-bromobenzyloxycarbonyl for tyrosine, 2-chlorobenzyloxycarbonyl forlysine, and tosyl for arginine. Arginine, asparagine, tyrosine, andselected proline and phenylalanine residues were double-coupled.Dimethylsulfoxide and anisole were included in all of the hydrogenfluoride cleavage solutions. Peptides were assessed by reverse-phaseHPLC and selected peptides were purified by this technique. Acetylationof the N-terminus of Tell to form Ac-T-11 was accomplished with aceticanhydride prior to cleavage from the resin. The mass of selectedpeptides was determined by fast atom bombardment mass spectrometry aspreviously described (Beer et al. 1992). T-6' was prepared by firstsynthesizing the peptide Phe-Leu-Leu-Arg-Asp by the automated method asdescribed in the previous paragraph and then manually adding thet-butoxycarbonyl racemic isoserine in dichloromethane anddimethylformamide (2:1) to the peptide on the resin using1-hydroxybenzotriazole to activate the isoserine andN,N'-diisopropylcarbodiimide to couple it to the peptide. The peptidewas then cleaved from the resin as described in the previous paragraph.

EXAMPLE 4 Rapid Whole Blood Assay for Determining Blockade of PlateletGlycoprotein IIb/IIIa Receptors

Blood (9 volumes) is drawn from a patient by syringe and placed in astandard blue-top tube containing sodium citrate (1 volume of 3.8%sodium citrate). Alternatively, blood may be drawn by vacutainerdirectly into a blue-top tube. The tube is then inverted to mix theanticoagulant with the whole blood. Then 50 μl of this mixture is addedto 50 μl of buffer (0.15M NaCl, 0.5 mM CaCl₂, 0.05M HEPES, pH 7.4) andto 5 μl of fibrinogen coated beads prepared by the method of Example 2.The mixture is rotated on a glass plate for about two minutes. IfGPIIb/IIIa receptors are blocked, the beads remain in suspension. If theGPIIb/IIIa receptors are not blocked, the platelets interact with thefibrinogen bound to the surface of the beads, resulting in clumping ofthe beads.

EXAMPLE 5 Rapid Whole Blood Assay for Determining Whether 80% ofGlycoprotein IIb/IIIa is Blocked

Blood (9 volumes) is drawn from a patient by syringe and placed intostandard blue-top tube containing sodium citrate (1 volume of 3.8%sodium citrate). Alternatively, blood may be drawn by Vacutainerdirectly into a blue-top tube. The tube is then inverted to mix theanticoagulant with the whole blood. Then 70 μl of this mixture is addedto an assay tube containing 100 μl of buffer (0.15M NaCl, 0.5 mM CaCl₂,0.01M HEPES, pH 7.4), 10 μl of well-mixed fibrinogen coated beadsprepared by the method of Example 1, and 5-10 pl ofisoSer-Phe-Leu-Leu-Arg-Asn (SEQ ID NO:3).

The assay tube is then placed on a rocker and rocked at 20 cpm at roomtemperature for two minutes. The tube is removed from the rocker, heldvertically to allow the blood to drain and viewed. If less than 80% ofGPIIb/IIIa receptor blockade has been achieved in the patient's blood,agglutinated beads are readily seen in the streak marking where theblood tilted back and forth. If greater than 80% of GPIIb/IIIa receptorblockade has been achieved in the patient's blood, the streak does notcontain bead agglutinates.

EXAMPLE 6 Correlation of Agglutination to GPIIb/IIIa Blockade

Purified peak I-2 fibrinogen is coupled to 3 μm, blue carboxylated beads(obtained from Polysciences, Inc.) using a water soluble carbodiimide(EDC) by the method of Example 1. The assay is conducted by adding to a12×75 mm glass test tube: 100 μl of buffer, 20 μl of beads (2.5%slurry), and 5 μl of isoSer-Phe-Leu-Leu-Arg-Asn. Blood is collected intocitrate anticoagulant and 70 μl of the whole blood is added to the testtube, which is then capped, placed on a rocking platform, and rocked at16 cycles/min. When normal blood was tested using 1 or 2 μM activatingpeptide, a 4+ agglutination endpoint was easily observed in all donors(n=10) by 2 min. Pretreating the blood with c7E3 Fab at 5 doses between0.8-3.0 μg/ml for about 10 minutes at 22° C. produced increasingGPIIb/IIIa receptor blockade and elimination of agglutination. Using 2μM activating peptide, the assay remained positive (i.e., 1-4+) at c7E3Fab concentrations of 0.8 (n=1), 1.2 (n=1), 1.5 (n=3), and 1.8 (n=2)μg/ml, corresponding to GPIIb/IIIa receptor blockade of 56%; 77%; 68,68, and 82%; and 66 and 73%. The assay became negative (i.e., O+) atc7E3 Fab concentrations of 1.2 (n=1), 1.5 (n=1), 1.8 (n=3) and 2.4 (n=2)μg/ml, corresponding to GPIIb/IIIa receptor blockade of 77%; 92%; 83, 88and 94%; and 88 and 89%. Aspirin (1 mM) and/or heparin (3.5 U/ml) didnot appreciably affect the results. The percentage of receptor blockadewas assayed by radiolabeled 7E3 binding as reported in Coller et al.,N.Y. Acad. Sci., 614:193 (1993).

EQUIVALENTS

Those skilled in the art will know, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerPheLeuLeuArgAsnProAsnAspLysTyrGluProPhe                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       SerPheLeuLeuArgAsnProAsnAspLysTyr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= ""xaa=isoserine""                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       XaaPheLeuLeuArgAsn                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       SerPheLeuLeuArgAsn                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= ""xaa=isoserine""                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       XaaPheLeuLeuArgAsnProAsnAspLysTyr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= ""xaa=isoserine""                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       XaaPheLeuLeuArgAsnProAsnAspLysTyrGluProPhe                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       SerPheLeuLeuArg                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= ""xaa=isoserine""                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       XaaPheLeuLeuArg                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Xaa=Gly(1-20)"                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       XaaArgGlyAsp                                                                  (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Xaa=Gly(9-11)"                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      XaaArgGlyAspPhe                                                               15                                                                            __________________________________________________________________________

What is claimed is:
 1. A method of determining whether an individual hasreduced ability to form platelet thrombi, comprising the steps of:a)obtaining a blood sample from the individual being assessed; b) mixingthe blood sample with 1) an anticoagulant; 2) sufficient buffer tomaintain the pH and salt concentration of the anticoagulated bloodwithin a range suitable for platelet aggregation; 3) a plateletGPIIb/IIIa receptor ligand immobilized on a solid surface and 4) aplatelet aggregation activator; c) agitating the mixture formed in b)for a period of time sufficient for unblocked platelet GPIIb/IIIareceptors to bind with the platelet GPIIb/IIIa receptor ligands on saidsolid surface; and d) assessing platelet-mediated agglutination in theagitated mixture, wherein the absence of agglutination indicates thatthe individual has reduced ability to form platelet thrombi.
 2. Themethod of claim 1 wherein the GPIIb/IIIa receptor ligand is selectedfrom the group consisting of fibrinogen, monoclonal antibody lOE5,monoclonal antibody c7E3, von Willebrand factor, fibronectin,vitronectin, and synthetic ligands having an arginine-glycine-asparticacid (RGD) GPIIb/IIIa binding sequence.
 3. The method of claim 1 whereinthe platelet aggregation activator is selected from the group ofadenosine diphosphate, collagen, ristocetin, botrocetin, epinephrine,arachidonic acid and its metabolites, platelet activating factor,plasmin, serotonin, vasopressin, tissue plasminogen activator,streptokinase and immune complexes.
 4. The method of claim 3 wherein thearachidonic acid metabolite comprises thromboxane A₂.
 5. The method ofclaim 1 wherein the solid surface comprises small glass or polymericbeads, the surface of said beads modified with a GPIIb/IIIa receptorligand.
 6. The method of claim 5 wherein the small polymeric beads havediameters from about 1 to about 6 μm.
 7. The method of claim 5 whereinthe small polymeric beads comprise carboxylated polystyrene orcarboxylated polyacrylonitrile beads.
 8. A method of determining thedegree of GPIIb/IIIa receptor blockade in a blood sample, comprising thesteps of:a) obtaining a blood sample from an individual being assessed;b) mixing the blood sample with 1) an anticoagulant; 2) sufficientbuffer to maintain the pH and salt concentration of the anticoagulatedblood within a range suitable for platelet aggregation; 3) a plateletGPIIb/IIIa receptor ligand immobilized on a solid surface and 4) aplatelet aggregation activator; c) agitating the mixture formed in b)for a period of time sufficient for unblocked platelet GPIIb/IIIareceptors to bind with the platelet GPIIb/IIIa receptor ligands on saidsolid surface; and d) assessing platelet-mediated agglutination in theagitated mixture, wherein the absence of agglutination indicates thatabout 80% or more of the GPIIb/IIIa receptors are blocked, and thepresence of normal agglutination indicates that less than about 50% ofthe GPIIb/IIIa receptors are blocked.
 9. The method of claim 8 whereinthe GPIIb/IIIa receptor ligand is selected from the group consisting offibrinogen, monoclonal antibody lOE5, monoclonal antibody c7E3, vonWillebrand factor, fibronectin, vitronectin, and synthetic ligandshaving an arginine-glycine-aspartic acid (RGD), or RGD functionalequivalent GPIIb/IIIa binding sequence.
 10. The method of claim 8wherein the platelet aggregation activator is selected from the group ofadenosine diphosphate, collagen, ristocetin, botrocetin, epinephrine,arachidonic acid and its metabolites, platelet activating factor,plasmin, serotonin, vasopressin, tissue plasminogen activator,streptokinase and immune complexes.
 11. The method of claim 10 whereinthe arachidonic acid metabolite comprises thromboxane A₂.
 12. The methodof claim 8 wherein the platelet aggregation activator comprises anN-terminal isoSer, 2,3-diaminopropanoate, or 2,3-dihydroxypropanoatethrombin receptor activating peptide.
 13. The method of claim 8 whereinthe solid surface comprises small glass or polymeric beads, the surfaceof said beads modified with a GPIIb/IIIa receptor ligand.
 14. The methodof claim 13 wherein the small polymeric beads have diameters from about1 to about 6 μm.
 15. The method of claim 13 wherein the small polymericbeads comprise carboxylated polystyrene or carboxylatedpolyacrylonitrile beads.
 16. A method of diagnosing Glanzmannthrombasthenia, other platelet function defects, or thrombocytopenia inan individual, comprising the steps of:a) obtaining a blood sample froman individual being assessed for Glanzmann thrombasthenia, otherplatelet function defects, or thrombocytopenia at a time when theindividual has not been administered an agent that blocks plateletGPIIb/IIIa receptors; b) mixing the blood sample with 1) ananticoagulant; 2) sufficient buffer to maintain the pH and saltconcentration of the anticoagulated blood within a range suitable forplatelet aggregation; and 3) a platelet GPIIb/IIIa receptor ligandimmobilized on a solid surface; c) agitating the mixture formed in b)for a period of time sufficient for unblocked platelet GPIIb/IIIareceptors to bind with the platelet GPIIb/IIIa receptor ligands on saidsolid surface; and d) assessing platelet-mediated agglutination in theagitated mixture, wherein the absence of agglutination is indicative ofGlanzmann thrombasthenia, other platelet function defects, orthrombocytopenia.
 17. The method of claim 16 wherein the GPIIb/IIIareceptor ligand is selected from the group consisting of fibrinogen,monoclonal antibody lOE5, monoclonal antibody c7E3, von Willebrandfactor, fibronectin, vitronectin, and synthetic ligands having anarginine-glycine-aspartic acid (RGD) or RGD functionally equivalentGPIIb/IIIa binding sequence.
 18. The method of claim 16 furthercomprising the addition of a platelet aggregation activator in step b).19. The method of claim 18 wherein the platelet aggregation activator isselected from the group of adenosine diphosphate, collagen, ristocetin,botrocetin, epinephrine, arachidonic acid and its metabolites, plateletactivating factor, plasmin, serotonin, vasopressin, tissue plasminogenactivator, streptokinase and immune complexes.
 20. The method of claim19 wherein the arachidonic acid metabolite comprises thromboxane A₂. 21.The method of claim 18 wherein the platelet aggregation activator is athrombin receptor activator.
 22. The method of claim 21 wherein thethrombin receptor activator is a thrombin receptor activating peptide.23. The method of claim 22 further comprising adding a plasmaaminopeptidase M inhibitor to the mixture of blood, anticoagulant,buffer, GPIIb/IIIa receptor ligand, and thrombin receptor activatingpeptide.
 24. The method of claim 22 wherein the thrombin receptoractivating peptide is selected from the group consisting of (SEQ ID NOS:1, 2, 4 and 7).
 25. The method of claim 24 further comprising adding aplasma aminopeptidase M inhibitor to the mixture of blood,anticoagulant, buffer, GPIIb/IIIa receptor ligand, and thrombin receptoractivating peptide.
 26. The method of claim 22 wherein the thrombinreceptor activating peptide comprises a peptide resistant toinactivation by plasma aminopeptidase M.
 27. The method of claim 26wherein the thrombin receptor activating peptide comprises an N-terminalisoSer, 2,3-diaminopropanoate, or 2,3-dihydroxypropanoate peptide. 28.The method of claim 27 wherein the thrombin receptor activating peptidecomprises at least 5 but no more than about 14 amino acids, wherein: R₁is selected from the group consisting of isoserine,2,3-diaminopropanoate or 2,3-dihydroxypropanoate; R₂ is any hydrophobicamino acid residue having a cyclic or aromatic group; R₃ is anyhydrophobic amino acid; R₄ is any hydrophobic amino acid; each of R₅ toR₁₄, if present, is an L-amino acid with the terminal amino acid being acarboxy or carboxamide, or a moiety that enhances cell penetratingability of the polypeptide, the cell penetrating moiety selected fromthe group consisting of lipids, steroids, long chain amines and sugars.29. The method of claim 28 wherein: R₂ is selected from the groupconsisting of phenylalanine, p-benzoylphenylalanine,p-fluorophenylalanine, and p-methylphenylalanine; R₃ is selected fromthe group consisting of leucine, phenylalanine, and alanine; R₄ isselected from the group consisting of leucine, and alanine; and R₅ isselected from the group consisting of arginine, and alanine.
 30. Themethod of claim 29 wherein R₆ is asparagine.
 31. The method of claim 27wherein the N-terminal isoSer peptide is selected from the groupconsisting of (SEQ ID NOS: 3, 5, 6, and 8).
 32. The method of claim 31wherein the N-terminal isoSer peptide comprisesisoSer-Phe-Leu-Leu-Arg-Asn (SEQ ID No: 3).
 33. The method of claim 16wherein the solid surface comprises small glass or polymeric beads, thesurface of said beads modified with a GPIIb/IIIa receptor ligand. 34.The method of claim 33 wherein the small polymeric beads have diametersfrom about 1 to about 6 μm.
 35. The method of claim 33 wherein the smallpolymeric beads comprise carboxylated polystyrene or carboxylatedpolyacrylonitrile beads.